Scientific and technical

Absence of salting out effects in forensic blood alcohol determination at various concentrations of sodium fluoride using semi-automated headspace gas chromatography
BA Miller*, SM Day, TE Vasquez and FM Evans California Department of Justice, Bureau of Forensic Services, Redding Regional Laboratory, 11745 Old Oregon Trail, Redding, CA 96002, USA Science & Justice 2004 44 73 -76 Original paper submitted August 2002 revised paper accepted 27 January 2004

Blood alcohol measurements determined by headspace gas chromatography have been challenged on the grounds that the presence of the preservative sodium fluoride in blood samples artificially increases headspace alcohol concentrations due to a salting out effect. Blood samples containing varying amounts of ethanol and sodium fluoride were tested using semi-automated headspace gas chromatography with n-propyl alcohol as the internal standard to assess the validity of this challenge. We find, in fact, that under these test conditions the measured alcohol levels are systematically depressed as the amount of sodium fluoride in the blood sample increases. The challenge thus has no basis. Les mesures du taux d'alcool dans le sang dCterminC par chromatographie en phase gazeuse par espace de t&te ont CtC contestCes sur la base de la prCsence du fluorure de sodium cornrne agent prCservateur dans les Cchantillons de sang qui augmenterait artificiellement les concentrations d'alcool dans l'espace de t&teB cause d'un effet de dCssalement (salting out). Des Cchantillons de sang contenant des quantitCs variables d'kthanol et de fluorure de sodium ont Ctk test& en utilisant une analyse par chromatographie en phase gazeuse et espace de t&te semi-automatisk, avec l'alcool n-propylique comme standard interne, pour determiner la validit6 de cette contestation. Nous trouvons en fait que dans ces conditions de test les taux d'alcool mesures sont systCmatiquement dCprimCs lorsque la quantitC de fluorure de sodium dans le sang augmente. La contestation est ainsi sans fondement.

Bestimmungen des Alkoholgehalts im Blut mittels HeadspaceGaschromatographie wurden angezweifelt, weil das zur Konservierung der Blutprobe zugegebene Natriumfluorid die Alkoholkonzentration in der Gasphase durch Aussalzen erhohen soll. Um die Stichhaltigkeit der Anfechtung zu iiberpriifen, wurden Blutproben mit unterschiedlichen Gehalten von Ethanol und Natriumfluorid mit halbautomatisierter HeadspaceGaschromatographie und n-Propanol als internen Standard untersucht. Tatsachlich finden wir aber unter den gewiihlten Testbedingungen eine systematische Absenkung der gemessenen Alkoholgehalte mit steigendem Gehalt von Natriumfluorid in den Blutproben. Die Anfechtung ist somit nicht begriindet. Las medidas de alcohol etflico en sangre por cromatografia de gases con espacio de cabeza han sido cuestionadas sobre la base de que el conservante fluoruro s6dico aiiadido a las muestras de sangre incrementa artificialmente las concentraciones de alcohol en el espacio de cabeza debido a un efecto salino. Se analizaron muestras de sangre conteniendo cantidades variables de etanol y fluoruro s6dico utilizando cromatografia de gases con espacio de cabeza semiautomitico y n-propil alcohol como estindar interno para valorar la validez de esta hip6tesis. Encontramos que , de hecho, bajo las condiciones de anilisis empleadas, 10s niveles de alcohol medidos eran sistemiticamente mas bajos a medida que la cantidad de fluoruro sbdico en la muestra de sangre se incrementaba. Por lo tanto dicha hipotesis carece de base.

*Author for correspondence O The Forensic Science Society 2004 Key words Forensic science, toxicology, alcohol, sodium fluoride, gas chromatography, salting out.

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BA Miller, SM Day, TE Vasquez and FM Evans Absence of salting out effects in forensic blood alcohol determination at various concentrations of NaF using semi-automated headspace GC

Blood samples collected for alcohol analysis typically include an added preservative such as sodium fluoride (NaF) [1,2]. Forensic laboratories using headspace gas chromatography for the analysis of ethanol in blood are sometimes challenged in court on the grounds that the concentration of ethanol in the headspace of a sample is artificially elevated due to the 'salting out' effect of NaF [3]. This challenge has also been raised in court cases where smaller than standard volumes of blood are drawn into the sample tube [4]. Salting out occurs when an inorganic salt is introduced into a miscible organiclaqueous liquid; water hydrates the salt ions and disrupts the weak hydrogen bonds between the water and the miscible organic liquid, resulting in a change in the partition coefficient between the vapor and liquid phases favoring an increase in the concentration of the organic in the vapor headspace of a sample [5,6]. The 'salting out' challenge stems from a statement made by AW Jones in 1983, 'With sodium fluoride as the blood anticoagulant at 2.0, 5.0, and 10.0 mglml, the concentration of ethanol in the equilibrated air phase rose by 3.2%, 5.4%, and 8.9%, respectively compared with heparinized blood' [7]. The salting out issue was later clarified in an exchange between AA Solanky [8] and Jones [9] in which the latter pointed out that his 1983 paper employed direct measurement of headspace ethanol rather than measurement using an internal standard such as npropanol as is standard forensic practice. The use of an internal standard such as n-propanol in blood alcohol analysis by headspace gas chromatography mitigates the salting out effect because both ethanol and the n-propanol standard are subject to the same salting out effect. Indeed, Jones has provided data indicating that increasing NaF concentration in blood samples with n-propanol as the internal standard resulted in an apparent decrease in measured ethanol levels, suggesting that n-propanol was salted out to a greater extent than ethanol [4,9]. Despite this clarification, the 'salting out' challenge continues to be made. We describe here experiments designed to determine the effects of NaF salting out on blood ethanol determination by semi-automated headspace gas chromatography under the standard analytical conditions used in this laboratory. Materials and methods Blood samples were drawn or deposited into 10 ml BectonDickinson (B-D) gray stoppered tubes containing 100 mg NaF and 20 mg potassium oxalate. A Hamilton digital dilutor (Hamilton #77325) is utilized to draw 250 pl of blood and 1.25 ml of n-propanol internal standard solution into the hand probe, resulting in a 1:6 dilution. The sample is then expelled into a 20 ml glass headspace vials with a butyl rubber stopper Table 1

and aluminum crimp cap seal. The vial is then placed into a Tekmar 7000 headspace autosampler tray and equilibrated to 65C for 13 minutes prior to automated headspace sample injection into the gas chromatograph (GC). Samples were run on a Hewlett Packard Model 5890 Series I1 Gas chromatograph with a flame ionization detector and a 6' X 118" ID stainless steel column (GP60180 Carbopak C, 0.2% Carbowax 1500). GC runs were isothermal at 100C and the detector is set at 200C; total run times were two minutes. Samples were run in duplicate. The standard deviation on ethanol measurements using this protocol is 0.0010 g1100 mL. Samples were run with known standards in accordance with the California Department of Justice blood alcohol method, which is calibrated using six repetitions of a laboratory prepared secondary alcohol standard and one repetition of a resolution standard. Known standards are aqueous solutions of ethanol prepared from USP absolute ethanol and distilled water. The value for the secondary alcohol standard is determined using direct oxidation with 0.0217N primary grade potassium dichrornate titrated with a solution of acidified ferrous ammonium sulfate using ferroin indicator. The value of the quality control alcohol standard is determined by GC analysis of 20 replicate samples referenced to the secondary alcohol standard. A resolution standard was made using reagent grade methanol, acetone, and isopropanol as well as USP absolute ethanol. The internal standard solution is reagent grade npropanol in distilled water at approximately 0.025% VN. Study subjects were selected from a pool of volunteers; subjects were informed of the nature of the study and its risks and were free to withdraw from the study at any time. All study subjects were required to sign a form of consent to participate in the study and provided answers to a medical questionnaire concerning current medications and general health. Subjects were given alcoholic beverages over a one hour period in an attempt to raise their blood alcohol concentrations to approximately 0.08% to 0.10% W N , the amount of alcohol provided was calculated based on the weight and sex of the individual using the SmithWidmark method [3]. A licensed medical technologist drew blood into a 10 cc syringe and deposited the blood into the standard B-D gray stoppered tubes. Samples were refrigerated at 4C as soon as possible after the blood draws. Results The effect of varying NaF concentration on the measurement of ethanol levels in three laboratory standards is shown in Table 1; values are given for replicate measurements and are rounded to

Detected Ethanol Levels in Laboratory Standards Containing Varying Amounts of NaF. Results are in g/100 ml. Duplicate determinations shown. QC - Quality control standards; SS - Secondary standards.
0 0.154, 0.153 10 0.154, 0.153 20 0.152, 0.152 30 0.152, 0.152 Mean 0.1 53 Std. Dev. 0.0009

Standard Sample

QC - 0.154

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BA Miller, SM Day, TE Vasquez and FM Evans Absence of salting out effects in forensic blood alcohol determination at various concentrations of NaF using semi-automated headspace GC

the nearest 0.001 g1100 mL. With all three standards, the measured amount of ethanol decreases slightly as the NaF concentration increases from zero to 30 mg/mL; regression analysis indicates an apparent decrease of 0.00006-0.00010 g1100 mL in measured ethanol per mg/mL increase in NaF. The differences among the measurements is small, however, and the averages of the eight measurements on each standard irrespective of NaF concentration differ from the independently determined standard value by no more than 0.001 gI100 mL with standard deviations of 0.0025 g/ 100 mL or less. Table 2 shows the results of testing on blood samples drawn from six subjects who had previously imbibed ethanol. Prior to the blood draws, the contents of 18 Becton-Dickinson tubes were reconstituted so as to contain potassium oxalate plus 0, 5,

or I0 mglml NaF after the addition of blood; 10 mg1mL represents the manufacturer's level. Blood was drawn from the subjects near the time of estimated peak blood alcohol concentration. As indicated in the table, blood alcohol levels determined from sample tubes containing 10 mg/mL NaF were equal to or lower than the levels detected in the sample tubes containing no NaF; the average decrease for the six samples was 0.0013 gI100 mL. The standard deviations on the ethanol measurements irrespective of NaF were all within 0.002 g1100 mL. The inverse relationship between NaF concentration and measured ethanol level is illustrated more dramatically by the data in Table 3. Blood was drawn from each of four subjects at two time points, first near the time of estimated peak blood

Table 2

Detected Ethanol Levels in Blood Samples Containing Varying Amounts of NaF. Results are in g/100ml. Duplicate determinations shown; standard deviations on duplicates < 0.0010. NaF (mglml) Subject 0 5 10 Mean Std. Dev.

Table 3

Detected Ethanol Levels in Blood Samples Containing Varying Amounts of NaF. Results are in g/100 ml. Values are averages of duplicate determinations; standard deviations on duplicates < 0.0010. NaF (mglml)

SubjecWSample

10 EtOH

20 EtOH

50

% difference

EtOH

Oh

difference

4b
Average % difference

0.0610

0.0595

2.5 2.8

0.0555

9.0 9.0

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BA Miller, SM Day, TE Vasquez and FM Evans Absence of salting out effects in forensic blood alcohol determination at various concentrations of NaF using semi-automated headspace GC

alcohol concentration (sample a) and then approximately 1.5 hours later (sample b). Samples were initially analyzed with NaF at manufacturer's levels (ca. 10 mg/mL). Then additional NaF was added to each sample based on the remaining volume of sample to raise the concentration successively to 20 mg/ml and 50 mg/ml NaF per tube; the tubes were opened each time additional NaF was added and mixed. For all eight samples, there is a clear trend for the measured ethanol levels to decrease with increasing NaF concentrations; the measured alcohol levels were reduced by an average 2.8% and 9.0% respectively in samples containing 20 mg/mL and 50 mgImL NaF relative to the levels found in the standard 10 mg/ mL tubes.

References
1 Dubowski KM. Alcohol Determination in the Clinical Laboratory. American Journal of Clinical Pathology 1980; 74(5):747-750.

Jones AW and Pounder DJ. Measuring Blood-Alcohol Concentration for Clinical and Forensic Purposes. In: Karch S, ed. Drug Abuse Handbook. Boca Raton: CRC Press, 1998: 327455.

3 4

California Drunk Driving Defense, 3rd ed.; Taylor, L.; West Group: 2001. Jones AW and Fransson M. Blood Analysis by Headspace Gas Chromatography; Does a Deficient Sample Volume Distort Ethanol Concentration? Medicine Science and the Law 2 m ; 43: 241-247.

Watts MT and McDonald OL. Effect of Biological Specimen Type on the Gas Chromatographic Analysis of Ethanol and Other Volatile Compounds. American Journal of Clinical Pathology 1987; 87: 79-85.

Discussion and conclusion This study shows that there is no artificial elevation of measured ethanol levels due to salting out effects when blood alcohol tubes containing NaF as a preservative are analyzed by a standard protocol employing headspace gas chromatography with npropanol as an internal standard. Indeed, the data provide experimental confirmation of Jones' observation that measured ethanol levels are in fact lower than the true ethanol level, the extent of the reduction depending on the concentration of NaF present [4,9]. This decline is a consequence of the more efficient salting out of n-propanol compared to ethanol due to the additional methyl group on the former [5,6]. Although it is possible that the results shown in Table 3 were affected by headspace loss occurring when the sample tubes were opened and closed, the observation of the same trend in the other two measurement series which involved no tube opening and closing suggests that headspace loss had little if any effect on the observed results.
These data demonstrate that the 'salting out' challenge to blood alcohol measurements determined by headspace gas chromatography with n-propanol as an internal standard is a bogus challenge. Indeed, under these analytical conditions, the presence of NaF in the blood collection tube depresses the measured ethanol value, resulting in the reporting of a value more beneficial to the defendant.

Watts MT and McDonald OL. The Effect of Sodium Chloride Concentration, Water Content, and Protein on the Gas Chromatographic Analysis of Ethanol in Plasma. American Journal of Clinical Pathology 1990; 93: 357-362.

Jones AW. Determination of IiquidJair partition coefficients for dilute solutions of ethanol in water, whole blood, and plasma. Journal of AnalyticalToxicology 1983 JulAug; 7(4): 193-7.

Solansky M.Effect of Diierent Concentrations of Sodium Fluoride on Blood Alcohol Determination by Headspace Gas Chromatography Using the Internal Standard Method. Journal of Analytical Toxicology 1994 Jan-Feb; 18: 63.

Jones AW. Salting-Out Effect of Sodium Fluoride and Its Influence on the Analysis of Ethanol by Headspace Gas Chromatography.Journal of Analytical Toxicology 1994 S e ~ t 18: 292-293. ;

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